WO2018158397A1 - Procédé d'élimination d'anticorps anti-aav présents dans une composition dérivée du sang - Google Patents

Procédé d'élimination d'anticorps anti-aav présents dans une composition dérivée du sang Download PDF

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Publication number
WO2018158397A1
WO2018158397A1 PCT/EP2018/055108 EP2018055108W WO2018158397A1 WO 2018158397 A1 WO2018158397 A1 WO 2018158397A1 EP 2018055108 W EP2018055108 W EP 2018055108W WO 2018158397 A1 WO2018158397 A1 WO 2018158397A1
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aav
aav8
blood
support
antibodies
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PCT/EP2018/055108
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English (en)
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Bérangère BERTIN
Carole MASURIER
Otto-Wilhelm Merten
Federico Mingozzi
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Genethon
INSERM (Institut National de la Santé et de la Recherche Médicale)
Sorbonne Université
Association Institut De Myologie
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Application filed by Genethon, INSERM (Institut National de la Santé et de la Recherche Médicale), Sorbonne Université, Association Institut De Myologie filed Critical Genethon
Priority to EP18708408.2A priority Critical patent/EP3589311A1/fr
Priority to US16/490,181 priority patent/US11376321B2/en
Publication of WO2018158397A1 publication Critical patent/WO2018158397A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/235—Adenoviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector

Definitions

  • the present invention relates to a method for removing undesired anti-AAV neutralizing antibodies, from a blood-derived composition.
  • AAV adeno-associated virus
  • anti-AAV neutralizing antibodies can completely prevent transduction of a target tissue, resulting in lack of efficacy, particularly when the vector is administered directly into the bloodstream (Masat, Pavani, Mingozzi, 2013, Discov Med. 2013, 15(85):379-89).
  • Anti-AAV NAbs are highly prevalent in humans, and the frequency of subjects with detectable titers can reach up to two thirds of the population.
  • the approach to the problem of preexisting humoral immunity to AAV so far has been the exclusion of seropositive subjects from enrollment in gene therapy clinical trials, but this solution is far from being optimal.
  • IVIG intravenous immunoglobulins
  • transient immunosuppression With respect to transient immunosuppression, one potential advantage when used in the context of AAV gene transfer is that, differently from organ transplant or autoimmune disease, the duration of the intervention in gene transfer would be relatively short, in the range of few hours (Murphy et al., 2008, Mol. Ther., 16(1):138-145) but one major limitation to the use of immunosuppression to achieve the complete eradication of anti-AAV antibodies is the lack of antigen specificity of the approach, which raises concerns over the risk of serious infections (Ginzler et al., 2012, Arthritis Res Ther, 14(1):R33) and malignancies .
  • the invention in a first aspect, relates to a method for removing anti-AAV antibodies, preferably anti- AAV neutralizing antibodies, from a blood-derived composition, comprising contacting said blood- derived composition with at least one support onto which one or more affinity ligand(s) specific of anti- AAV antibodies is bound.
  • the method may be implemented in vitro or extracorporeally (i.e. ex vivo), as will be provided in details below.
  • said one or more affinity ligand is an antigenic affinity ligand such as an AAV particle (either full or empty), an AAV capsid protein (or a fragment thereof), or a capsid peptide mimic of one or more serotypes.
  • the AAV particle is an empty AAV particle.
  • the composition may be contacted with more than one support, wherein each support may have different affinity ligands specific of different AAV serotypes bound thereon.
  • the affinity ligand may be specific of an AAV serotype which is the serotype of an AAV gene therapy vector intended to be used in a subject in need thereof, and wherein the blood-derived composition is either from said subject, or is intended to be administered to said subject.
  • Illustrative affinity ligands include those specific of serotype AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 (such as -cylO or -rhlO), -11, -rh74 or engineered AAV capsid variants such as AAV-2i8, AAV2G9, -LK3, -DJ, and -Anc80.
  • an affinity ligand specific of AAV8 is used.
  • the method of the invention comprises contacting the blood-derived composition with a support onto which are bound a first affinity ligand specific of a first AAV serotype (for example specific of the AAV2 serotype) and a second affinity ligand specific of a second AAV serotype (for example specific of the AAV8 serotype).
  • a first affinity ligand specific of a first AAV serotype for example specific of the AAV2 serotype
  • a second affinity ligand specific of a second AAV serotype for example specific of the AAV8 serotype.
  • more than two affinity ligands, providing specificity for more than two AAV serotypes are bound onto the support.
  • the method of the invention comprises contacting the blood-derived composition with at least:
  • an affinity ligand specific of a second AAV serotype for example specific of the AAV8 serotype
  • a set of affinity ligand specific of said second AAV serotype are bound.
  • each additional support may comprises bound thereon at least one additional affinity ligand specific for at least one additional AAV serotype.
  • two or more supports are used, each comprising one or more affinity ligand(s) bound thereon, specific of one or more AAV serotypes.
  • a first support may be used onto which an affinity ligand specific of a first AAV serotype is bound
  • a second support is used onto which an affinity ligand specific of a second AAV serotype and an affinity ligand specific of a third AAV serotype are bound.
  • a first support may be used onto which an affinity ligand specific of a first AAV serotype and an affinity ligand specific of a second AAV serotype are bound, and a second support is used onto which an affinity ligand specific of a third AAV serotype (and optionally one or more affinity ligand(s) specific of one or more additional AAV serotype(s), respectively) is bound.
  • the blood-derived composition submitted to the method of the invention may be, in particular whole blood, blood plasma, blood plasma fractions, blood plasma precipitate (e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), blood plasma supernatant (e.g., cryosupematant, ethanol supernatant or polyethylene glycol supernatant), solvent/detergent (SD) plasma, platelets, intravenous immunoglobulin (IVIG), IgM, purified coagulation factor concentrate, fibrinogen concentrate, or various other compositions which are derived from human or animal.
  • the blood-derived composition is whole blood or an IVIG composition.
  • the blood-derived composition is loaded several times onto the same support (for example 2, 3, 4 or 5 times or more than 5 times) and/or the composition is loaded on several different columns serially arranged, either grafted with the same or different affinity ligand(s).
  • Another aspect of the invention relates to a composition obtainable by applying the method described above on a blood-derived composition.
  • the composition of the invention is therefore a blood-derived composition deprived in AAV antibodies.
  • the blood-derived composition may in particular be whole blood, blood plasma, blood plasma fractions, blood plasma precipitate (e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), blood plasma supernatant (e.g., cryosupematant, ethanol supernatant or polyethylene glycol supernatant), solvent/detergent (SD) plasma, platelets, intravenous immunoglobulin (IVIG), IgM, purified coagulation factor concentrate, fibrinogen concentrate, or various other compositions which are derived from human or animal.
  • the blood-derived composition is whole blood or an IVIG composition.
  • a further aspect of the invention relates to a support onto which one or more affinity ligand specific of one or more anti-AAV antibody, respectively, is grafted.
  • the grafted affinity ligand(s) may be an antigenic affinity ligand such as an AAV particle (either empty or full), an AAV capsid protein, or fragments thereof, or a capsid peptide mimic of one or more serotypes, more particularly an AAV particle, even more particularly an empty AAV particle.
  • the support may in particular be a poros or sepharose support, such as a sepharose support grafted with the affinity ligand via a NHS functional group.
  • the support according to the invention is grafted with
  • a first affinity ligand specific of a first AAV serotype such as (empty or full) AAV8 particles or AAV8 capsid proteins
  • a second affinity ligand specific of a second AAV serotype such as (empty or full) AAV2 particles or AAV2 capsid proteins, or
  • the invention further relates, in another aspect, to an AAV vector for use in a method for the treatment of a disease by gene therapy,
  • the AAV vector comprises a therapeutic gene of interest appropriate for the treatment of said disease
  • said AAV vector is of a given serotype
  • said AAV vector is for administration to a subject in need thereof after administration to said subject of a blood-derived composition which has been processed according to the method described above to remove anti-AAV antibodies specific of said given serotype from said composition.
  • the blood-derived composition is whole blood or wherein the subject had previously undergone plasmapheresis and the blood-derived composition is an IVIG composition.
  • Figure 1 Percentage of retained anti-AAV8 antibodies using POROS affinity columns to which 3.7x10 11 vg of AAV8 have been grafted.
  • the grafted AAV particles were either crosslinked by a formaldehyde treatment or not.
  • x-axis indicates the fractions (Al - B8: loading of the column followed by washing; B7 - C2: elution using a citrate buffer step; C3 - CI 2: reconditioning of the column);
  • z- axis ng of anti-AAV8.
  • FIG. 7 A. Removal of anti-AAV8 antibodies from IVIG and plasma using a 5ml AAV8-NHS- sepharose column grafted with 6x10 13 vg AAV8. In the first 4 runs, 2 different IVIG concentrations (36 and 72mg) were loaded to the column. In all subsequent runs, different volumes of three different human plasma samples with low (titer 1/3), medium (titer 1/270), and high (titer 1/7290) anti-AAV8 antibody titers were loaded and the retention of anti-AAV8 antibodies was evaluated by ELISA. B.
  • Figure 8 Comparison of two columns grafted with a low or a high amount of empty AAV8 capsids (2xl0 12 and 3.2xl0 13 , respectively) for removal of anti-AAV8 antibodies from a high tittering human plasma sample (1/7290) loaded at 2-3 different volumes. The retention of anti-AAV8 antibodies was assessed by ELISA.
  • FIG. 9 Removal of anti-AAV8 antibodies (A) from the human serum sample 'DRI' by passage over an AAV8-NHS column (5ml, grafted with l .lxl0 13 vg) and evaluation of the retention/non-retention of total human immunoglobulin (B). Note: Loading and washing: fractions A1-B5; elution with citrate buffer: B4-C5.
  • the present invention relates to a method for removing anti-AAV antibodies, preferably anti-AAV neutralizing antibodies (NAbs), from a blood-derived composition, comprising contacting said blood- derived composition with at least one support on which one or more affinity ligand(s) specific of anti- AAV antibodies is bound.
  • NAbs anti-AAV neutralizing antibodies
  • affinity ligand specific of an AAV serotype and “affinity ligand that specifically binds to anti-AAV antibodies directed toward an AAV serotype” are used interchangeably.
  • the anti-AAV-depleted compositions obtained according to the method of the present invention possess markedly reduced anti-AAV Ab titers, especially NAb titers. These low levels of anti-AAV NAbs allow for improved AAV-based gene therapy.
  • blood-derived compositions treated according to the method of the present invention have advantages in the field of AAV-mediated gene therapy. Administration of the resulting purified blood-derived compositions allow the further systemic administration of AAV vectors for gene therapy without having to resort to conventional plasmapheresis, which results in total removal of circulating immunoglobulins leading to immunodeficiency requiring supplementation with purified intravenous immunoglobulins (IVIG).
  • IVIG purified intravenous immunoglobulins
  • the present invention may be implemented on a blood-derived composition such as an IVIG preparation that may contain anti-AAV antibodies therein, thereby preventing the reintroduction of further anti-AAV antibodies to a patient in need of an AAV-based gene therapy.
  • a blood-derived composition such as an IVIG preparation that may contain anti-AAV antibodies therein, thereby preventing the reintroduction of further anti-AAV antibodies to a patient in need of an AAV-based gene therapy.
  • blood-derived compositions and “blood compositions” are used interchangeably and are meant to include whole blood, blood plasma, blood plasma fractions, blood plasma precipitate (e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), blood plasma supernatant (e.g., cryo-supernatant, ethanol supernatant or polyethylene glycol supernatant), solvent/detergent (SD) plasma, platelets, intravenous immunoglobulin (IVIG), IgM, purified immunoglobulins, or various other compositions which are derived from human or animal.
  • blood plasma precipitate e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate
  • blood plasma supernatant e.g., cryo-supernatant, ethanol supernatant or polyethylene glycol supernatant
  • solvent/detergent (SD) plasma platelets
  • IVIG intravenous immunoglobulin
  • IgM purified immunoglobulins
  • AAV is a small, nonenveloped virus of the parvovirus family that packages a single-stranded linear DNA genome, approximately 5 kb long.
  • AAV vectors are increasingly used for in vivo gene therapy thanks to their wide range of host cells, including non-dividing cells, and to the fact that AAVs have not been associated with any human or animal disease.
  • anti-AAV neutralizing antibodies are highly prevalent in humans, and the frequency of subjects with detectable titers can reach up to two thirds of the population.
  • the present invention aims at removing anti-AAV antibodies from a blood-derived composition, in order to improve AAV-mediated therapy.
  • Anti-AAV antibodies that can be removed include antibodies to any AAV vector that could be used in AAV- mediated therapy, such as antibodies to any naturally or non-naturally occurring AAV serotype.
  • a "serotype" is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
  • a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest.
  • AAV includes various naturally and non-naturally (e.g. hybrid, chimera or shuffled serotypes) occurring serotypes.
  • Such non-limiting serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 (such as -cylO or -rhlO), -11, -rh74 or engineered AAV capsid variants such as AAV-2i8, AAV2G9, -LK3, - DJ, and -Anc80.
  • AAV2G9 serotypes include those disclosed in EP2292779, EP1310571 and US7906111.
  • other AAV serotypes include those obtained by shuffling, as described in Koerber et al. (Molecular Therapy (2008), 16(10), 1703-1709), peptide insertion (e.g. Deverman et al., Nat Biotechnol (2016), 34(2), 204- 209), or rational capsid design (reviewed in Biining et al., Curr Opin Pharmacol (2015), 24, 94-104).
  • serotypes include AAV with capsid sequence modifications that have not been fully characterized as being a distinct serotype, and may in fact actually constitute a subgroup or variant of a known serotype.
  • a support is used onto which an affinity ligand specific of an anti-AAV antibody is bound (or "grafted”, as used interchangeably herein with “bound”, “bind” and declinations thereof when referring to the binding of a ligand to a support).
  • ligands may be any ligand that is either specifically recognized and bound by the anti-AAV antibody, or that may specifically recognize or bind the anti-AAV antibody.
  • the term "specifically” used with respect to an affinity ligand denotes the ability of said affinity ligand to recognize a motif, sequence, or structure of its binding partner and to interact with said binding partner by spatial complementarity.
  • An affinity ligand according to the invention "preferentially binds" to its target, meaning that it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an affinity ligand that preferentially binds to an anti-AAV8 antibody is an affinity ligand that binds this antibody with greater affinity, avidity, more readily, and/or with greater duration than it binds to antibodies different from an anti-AAV8 antibody. It should be understood by reading this definition that, for example, an affinity ligand that specifically or preferentially binds to a first target may or may not preferentially bind to a second target. As such, "preferential binding" does not necessarily require (although it can include) exclusive binding.
  • the affinity ligand may be, for example, an antigen recognized by the anti-AAV antibody.
  • Illustrative antigens that may be implemented in the present invention include AAV particles (e.g. genome-containing or empty AAV particles), AAV capsid proteins or fragments thereof comprising an epitope recognized by an anti-AAV antibody (such as peptide fragments of AAV capsid proteins exposed at the surface of the AAV particle), or peptides mimics of the epitope recognized by the anti-AAV antibody intended to be removed.
  • the affinity ligand may also be selected from molecules that specifically recognize the anti-AAV antibody such as aptamers derived against an anti-AAV antibody or immunoglobulins that are produced for specifically recognizing an anti-AAV antibody.
  • an "AAV particle” which is bound on a support is either a genome- containing AAV particle (or “genome-containing AAV capsid” or “full AAV particle” or “full particle”) or an empty AAV particle (otherwise referred to as an "empty AAV capsid” or "empty AAV particle”).
  • the affinity ligand is a genome-containing AAV particle, an empty AAV particle, or a combination of both a genome-containing AAV particle and an empty AAV particle.
  • the affinity ligand is an empty AAV particle.
  • the affinity ligand comprises a combination of empty and full particles, with an empty: full particle ratio comprised between 1 :100 and 100: 1, in particular between 1 : 10 and 10: 1, in particular between 1 :5 and 5: 1, in particular between 1 :3 and 3:1, such as a 1 : 1 ratio.
  • the AAV particle bound to a support is an empty or non-infectious AAV particle, which is advantageous in term of safety and with respect to the possible increased antibody binding capacity.
  • the non- infectious AAV particle is an AAV particle (either full or empty) which lacks the vpl protein, such as a capsid comprising only vp2 and/or vp3 capsid proteins, in particular an AAV particle whose capsid comprises only the vp3 capsid protein.
  • the AAV particle is an AAV2-derived particle.
  • the AAV particle capsid comprises all AAV vp capsid proteins, i.e. AAV vpl, vp2 and vp3 capsid proteins.
  • the AAV particle is an empty AAV capsid, preferably an empty AAV capsid comprising all AAV vp capsid proteins, i.e. AAV vpl, vp2 and vp3 capsid proteins.
  • AAV particles such as empty AAV particles or genome containing AAV particles, including AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74, AAV-2i8, AAV2G9, -LK3, -DJ, and Anc80, can be constructed using recombinant techniques that are known to the skilled artisan.
  • Full AAV particles can be produced by different means by a person skilled in the art. An illustrative method of production is detailed hereinafter.
  • Full AAV particles can be produced by transfection of, for instance, but not exclusively, HEK293 or HEK293T cells using a bi- or tri-plasmid transfection approach, in which the plasmids provide AAV helper functions (rep, cap), helper virus helper functions (in most of the cases of adenovirus 5, but not only) as well as a recombinant rAAV vector construct. 48-72h post-transfection, AAV particles may be harvested via cell lysis.
  • More advanced production methods include the use of stable cell lines, mainly based on HeLa cells, which contain the rep/cap functions of AAV as well as the recombinant rAAV vector construct.
  • the cells are infected with adenovirus 5 (or another virus providing helper virus function) and 72h post-infection, AAV particles may be harvested via cell lysis.
  • adenovirus 5 or another virus providing helper virus function
  • 72h post-infection AAV particles may be harvested via cell lysis.
  • the herpes simplex virus/BHK system and the baculovirus/insect cell system. The most recent versions of both systems are based on co-infection of the cells with two different viruses, one providing AAV helper functions rep and cap and the second one the recombinant rAAV vector construct.
  • helper virus functions are provided by the herpes simplex virus and the baculovirus, respectively.
  • AAV vector particles are harvested via cell lysis.
  • the reviews by Merten et al. (Gene Ther 2005, 12 Suppl 1, S51-61; Pharma. Bioproc. 2014, 2(2): 183-203 and 2(3), 237-251) are recommended.
  • the production of empty AAV vector particles is often an undesired by-product of the production of all AAV vector production technologies and can be separated from full AAV particles by ultracentrifugation.
  • the affinity ligand which is bound to the support is an antigenic affinity ligand (for example an AAV particle (either full or empty as defined above) an AAV capsid protein or fragments thereof, or capsid peptide mimics) from a serotype that reacts with antibodies present in the subject to be treated with AAV-based gene therapy.
  • the antigenic affinity ligand bound to the support is from a serotype corresponding to the serotype of the AAV vector that will be used as a gene therapy vector in the subject to be treated, independently of whether antibodies to this serotype were detected in said subject beforehand.
  • the antigenic affinity ligand bound on the support is from a serotype different to that of the AAV vector that will be used as gene therapy vector, but is a serotype that cross-reacts with said serotype of the AAV vector that will be used as gene therapy vector.
  • AAV2 particles or AAV2 capsid proteins may be bound to the support for the purpose of removing anti-AAV8 antibodies from a blood-derived composition, due to cross-reactivity of anti-AAV8 antibodies with AAV2.
  • Other cross-reactivities were described and are known to the prior art, such as from Boutin et al., Hum Gene Ther 2010, Jun; 21(6):704-12; Calcedo et al., J Infect Dis.
  • the antigenic affinity ligands specific of one or more serotype(s) are bound to the at least one support used in the method of the present invention.
  • AAV particles either full or empty
  • AAV capsid proteins or fragments thereof
  • capsid peptide mimics of more than one serotype may be bound on the same support, therefore providing a "set of antigenic affinity ligand" (e.g.
  • each support may comprise bound thereon antigenic affinity ligands of only one serotype, or may comprise bound thereon a set of affinity ligands, for example a set of AAV particles (either full or empty) of different serotypes, or a set of AAV capsid proteins (or fragments thereof) of different serotypes, or a set of capsid peptide mimic of different serotypes. Any combination of serotypes can be envisioned.
  • the method of the invention may implement antigenic affinity ligands derived from any combination of the specific serotypes AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVl 1, RhlO, Rh74, AAV-2i8, AAV2G9, -LK3, -DJ, and Anc80.
  • a first antigenic affinity ligand specific of the AAV2 serotype is combined to any other serotype, such as any of the serotypes listed in the preceding sentence.
  • a set of AAV2 and AAV8 particles, or a set of AAV2 and AAV8 capsid proteins is bound on the same support.
  • the at least one support comprises one support on which is bound AAV2 particles or AAV2 capsid proteins, and another support on which is bound AAV8 particles or AAV8 capsid proteins.
  • the support may be a molecularly imprinted polymer having affinity to one or more anti-AAV antibody.
  • the support of the invention may correspond to any kind of support onto which an affinity ligand can be bound, either covalently or non-covalently.
  • the support may correspond to any type of support as above, such as a compressible (such as a compressible smooth gel, e.g. sepharose) or noncompressible support (such as a robust incompressible high porosity support), in particular a sepharose, sephadex, agarose, cellulose, modified cellulose, CPG, poros or monolith support.
  • a compressible such as a compressible smooth gel, e.g. sepharose
  • noncompressible support such as a robust incompressible high porosity support
  • the support is a sepharose support.
  • the attachment need not be covalent, but is at least of sufficient permanence to withstand any separation techniques (including washes and elution) that may be part of the method of the present invention.
  • attachment to the support is via a covalent bond.
  • the affinity ligand is covalently linked to the support.
  • the affinity ligand which is covalently linked to the support is an antigenic affinity ligand, such as an AAV particle (either full or empty), an AAV capsid protein (or fragment thereof), or a capsid peptide mimic.
  • the antigenic affinity ligand covalently bound to the support is an AAV particle (either full or empty), such as an AAV particle selected in the groups consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74, AAV-2i8, AAV2G9, -LK3, -DJ, and Anc80.
  • the support may be, for example, a chomatographic column, particles or beads, Monoliths, a membrane or a filter.
  • the support may further be a hollow fiber cartridge.
  • the support may be an activated support, comprising functional groups allowing covalent chemical conjugation of the affinity ligand.
  • activated supports include, without limitation cyanogen bromide (CNBr)-activated supports, N-hydroxysuccinimide- activated supports, carbonyl diimidazole (CDI)-activated supports, and the like.
  • coupling materials that may be used as described herein include, for example, CNBr Sepharose Fast Flow, NHS Sepharose Fast Flow, Epoxy Sepharose 6B, Thiol Sepharose Fast Flow, EAH Sepharose Fast Flow, Epoxy Poros EP, Aldehyde Poros AL, Epoxy Poros EP, Hydroxylated Poros OH and CDI and epoxy Monolithic materials.
  • the support is a NHS Sepharose support. Coupling onto such supports is well known in the art, and is typically done following the manufacturer's instructions.
  • a protein cross-linking compound such as formaldehyde may be used.
  • Such a cross-linking compound may in particular be implemented on supports that activated with more labile functional groups.
  • the support functional groups are epoxy groups, and formaldehyde is used to cross-link the AAV particles or the AAV capsid proteins used in the practice of the present invention.
  • native, non-cross-linked AAV particles or AAV capsid proteins are implemented in the present invention.
  • an antibody specific of an AAV serotype is coupled to the support, then a preparation of the corresponding AAV serotype particle is provided, wherein the AAV particle binds to the antibody.
  • covalent linking between the antibody bound to the support and the AAV particle is implemented to obtain a support able to remove anti-AAV antibodies from a blood-derived composition.
  • the N-hydroxy succinimide (NHS) chemistry is implemented for covalently linking the affinity ligand.
  • the support is a support suitable for clinical use, i.e. a support complying with regulatory safety provisions for devices to be used in purification/preparation process of biopharmaceuticals for animal and human use.
  • the affinity ligand density (e.g. AAV particle density or AAV capsid protein density, or capsid peptide mimic density) on the support may vary to a large extent.
  • AAV particle density or AAV capsid protein density, or capsid peptide mimic density on the support may vary to a large extent.
  • AAV particle density or AAV capsid protein density, or capsid peptide mimic density on the support may vary to a large extent.
  • AAV particle density or AAV capsid protein density, or capsid peptide mimic density may vary to a large extent.
  • AAV particle density e.g. AAV particle density or AAV capsid protein density, or capsid peptide mimic density
  • capsid peptide mimic density on the support may vary to a large extent.
  • 9x10 11 vg to 9x10 13 vg AAV particles may be grafted, such as 1 x 10 13 vg, 2 x 10 13 vg, 3 x 10 13 v
  • the flow rate of used for conducting the present method is of between 0.01 and 1 ml/min, such as between 0.05 and 0.7 ml/min, for example between 0.1 and 0.5 ml/min (or 0.02 and 0.1 CV/min, wherein “CV” stands for "column volume”). In a particular embodiment, the flow rate is of about 0.1 ml/min (or 0.02 CV/min).
  • the present invention relates to a support onto which an affinity ligand specific of anti-AAV antibodies is grafted, such as an antigenic affinity ligand like AAV particles (such as full or empty particles), AAV capsid proteins or fragments thereof, or capsid peptide mimics.
  • the support may correspond to any type of support as described above, such as a compressible or noncompressible support, in particular a sepharose, sephadex, agarose, cellulose, modified cellulose, CPG, poros or monolith support.
  • the support is a sepharose support.
  • the support is grafted via a NHS functional group.
  • the support is grafted with an antigenic affinity ligand AAV particles or AAV capsid proteins of the any serotype, such as any naturally or non-naturally occurring (such as hybrid, chimeric, shuffled serotypes) serotype, for example with an AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVl l, RhlO, Rh74, AAV-2i8, AAV2G9, -LK3, -DJ and Anc80.
  • the support is grafted with AAV8 particles or AAV8 capsid proteins, or with AAV2 particles or AAV2 capsid proteins.
  • the support is grafted with AAV2 and AAV8 particles, or with AAV2 and AAV8 capsid proteins.
  • the invention also relates to an extracorporeal device through which a patient's blood or plasma can be circulated prior to being returned to the patient, said device comprising a support according to the invention.
  • binding of the anti-AAV antibodies to the affinity ligand(s) that specifically bind to anti-AAV antibodies removes the anti-AAV antibody molecules from the patient's blood or plasma, thereby allowing further treatment of the patient with AAV-based gene therapy.
  • anti-AAV antibodies such as anti-AAV NAbs are removed or depleted from a blood-derived composition.
  • total depletion of anti-AAV antibodies, in particular NAbs is not required, as long as the reduction in antibody titers allows an improvement in AAV vector transduction when AAV-based gene therapy is carried out after implementation of the above method.
  • antibody titer reduction may be of at least 1 log, 2 logs, 3 log, or at least 4 logs.
  • an anti-AAV neutralizing antibody titer of 1 :3,160 can be reduced to 1 : 1 (negative titer).
  • the blood-derived composition is passed once or several times through the at least one support described above.
  • the support may be serially arranged.
  • the same column is used for more than one retention cycle (either for the same blood-derived composition, or different compositions, preferably for the same).
  • composition of the invention relates to a composition obtainable by applying the method described above on a blood-derived composition.
  • the composition of the invention is therefore a blood- derived composition with reduced amounts (or titers) in AAV antibodies.
  • the anti-AAV antibody presence or content in the blood-derived composition is determined before applying the method of the present invention.
  • the presence or content of anti- AAV antibodies is not determined before application of the method of the present invention.
  • the composition of the present invention is obtainable from a blood-derived composition that contains anti-AAV antibodies. The resulting composition is thus a composition with reduced amounts (or titers) of anti-AAV antibodies as compared to the blood-derived composition before application of the method of the invention.
  • the blood-derived composition may in particular be whole blood, blood plasma, blood plasma fractions, blood plasma precipitate (e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), blood plasma supernatant (e.g., cryosupematant, ethanol supernatant or polyethylene glycol supernatant), solvent/detergent (SD) plasma, platelets, intravenous immunoglobulin (IVIG), IgM, purified coagulation factor concentrate, fibrinogen concentrate, or various other compositions which are derived from human or animal.
  • the blood-derived composition is whole blood or an IVIG composition.
  • the present invention also provides a method for improving transduction efficiency of an AAV gene therapy vector.
  • a number of embodiments are envisioned in this regard and are encompassed by the present invention.
  • the above method for removing anti-AAV (neutralizing) antibodies is applied to the whole blood of a subject who is a future receiver of an AAV-based gene therapy, thereby reducing anti-AAV (N)Abs titers in said whole blood.
  • the blood of the subject is moved from the subject along a pathway comprising the at least one support described above, and the anti-AAV (N)Ab-depleted blood is returned to the subject's internal circulation.
  • the subject's blood may be subjected to classical plasmapheresis, thereby non- specifically removing all or almost all antibodies from said blood, including anti-AAV (N)Abs.
  • an IVIG composition is submitted to the method for removing anti-AAV (N)Abs according to the invention, and this processed IVIG composition is then administered to the subject who was previously subjected to plasmapheresis, thereby preventing the IVIG composition from reintroducing anti-AAV (N)Abs into the subject's circulation.
  • the subject's blood is subjected to one or more cycles of anti-AAV antibody depletion according to the present invention, such as to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 cycles of anti-AAV antibody depletion (be it according to the method of the present invention, using one or more supports grafted with one or more affinity ligands as defined above, or using classical plasmapheresis followed by the injection of an anti- AAV-depleted IVIG composition).
  • the subject treated as provided in the preceding paragraph is then administered with the AAV gene therapy vector, comprising a therapeutic gene of interest selected for treating the subject's condition.
  • the AAV gene therapy vector is administered as soon as possible after the subject has been treated as provided above.
  • the subject is treated before anti-AAV titers raise again.
  • the subject treated as provided in the preceding paragraph is administered with an immunosuppressive compound before administration of the AAV gene therapy vector.
  • Illustrative immunosuppressive compounds include, without limitation, rituximab, Ocrelizumab, bortezomib, ibrutinib, ciclosporin A, calcineurin inhibitors, rapamycin, corticosteroids, mycophenolate mofetil, atacicept, baff inhibitors, BLyS and APRIL inhibitors
  • the present invention thus also relates to an AAV vector for use in a method for the treatment of a disease by gene therapy,
  • the AAV vector comprises a therapeutic gene of interest appropriate for the treatment of said disease
  • said AAV vector is of a given serotype, wherein said AAV vector is for administration to a subject in need thereof, after administration to said subject of a blood-derived composition which has been processed according to the method for removing anti-AAV antibodies described above, to remove anti-AAV antibodies specific of said given serotype from said composition.
  • the invention also relates to a method for the treatment of disease in a subject in need thereof by gene therapy, wherein the disease is treated by administering to said subject an effective amount of an AAV vector comprising a therapeutic gene appropriate for treating said disease, wherein
  • said AAV vector is of a given serotype
  • said AAV vector is for administration to said subject, after administration to said subject of a blood- derived composition which has been processed according to the method for removing anti-AAV antibodies described above, to remove anti-AAV antibodies specific of said given serotype from said composition.
  • the present invention may generally be applied for therapy of any disease that may be treated by expression of a therapeutic gene in a cell or tissue of a subject mediated by an AAV vector.
  • proliferative diseases cancers, tumors, dysplasias, etc.
  • infectious diseases include, for example, proliferative diseases (cancers, tumors, dysplasias, etc.), infectious diseases; viral diseases (induced, e.g., by the Hepatitis B or C viruses, HIV, herpes, retroviruses, etc.); genetic diseases (cystic fibrosis, dystroglycanopathies, myopathies such as Duchenne Muscular Myopathy; myotubular myopathy; hemophilias; diabetes; amyotrophic lateral sclerosis, motoneurones diseases such as spinal muscular atrophy, spinobulbar muscular atrophy, or Charcot-Marie-Tooth disease; arthritis; cardiovascular diseases (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), or neurological diseases (psychiatric diseases, neurodegenerative diseases such as Parkinson's or Alzheimer's, Huntington's disease addictions (e.g., to tobacco,
  • Sf9 cells (Gibco) were grown in suspension culture at 27°C in SFM900III medium (Invitrogen) using 1L Corning Erlenmeyer Flasks.
  • the baculovirus system used was the system as published by Smith et al. (Mol. Ther., 2009, 17(11), 1888-1896).
  • Baculoviruses were generated according to the guidelines of the Bac-to-Bac protocol and were amplified in suspension cultures of Sf9 cells in 250mL Erlenmeyer Flasks.
  • rAAV production were performed by dual infection of baculoviuses harboring the recombinant AAV genome (ySGC) and AAV rep/cap genes, each at an MOI of 0.05 (PFU titer) in 70mL of Sf9 cell culture seeded at 10 6 cells/mL in 250mL Erlenmeyer Flasks. At 96h post-infection, lmL of the total culture was recovered for direct quantification of rAAV production prior to purification,
  • the empty AAV8 vector particles were prepared according to Ayuso et al. Gene Therapy 2010.
  • a quantitative PCR assay was performed directly on the total culture samples or purified rAAV samples to determine the rAAV titer (viral genome copies per ml of culture).
  • Viral DNA was extracted directly from the bulk or from purified samples using the MagNA Pure DNA and viral NA small volume kit
  • Probe 5 ' -CAGAATCAACAGTTTCAG-3 ' [5']6-FAM[3']MGB-NFQ probe (SEQ ID NO:3).
  • Purified AAV samples were migrated on a SDS-PAGE (NuPAGE Novex 1.5mm 4-12% gel from INVITROGEN) by loading 1-10 ⁇ 1 of prepared sample per lane (most commonly: 2 ⁇ 1 per lane).
  • fixation solution was replaced by 60mL of SYPRO Ruby stain protein.
  • the box was closed and protected from the light (in alu paper) and slowly agitated overnight.
  • the gel was washed 2 times in water. In the Gbox, the gel was exposed to 200msec and a picture is taken.
  • the Licor software "image studio lite" was used to analyze and quantify the signal of VP3 in each sample. The sample concentration/titer was expressed as capsid equivalents per ml.
  • AVB sepharose (GE HealthCare) - immune-affinity based retention of AAV particles:
  • the column was further incubated with or without formaldehyde.
  • the incubation with formaldehyde was chosen for increasing the stability of graft/reducing AAV particle leakage.
  • optimal flow rate was determined to be of 0.1 mL/min (0.02 CV/min), which was the flow rate that allowed the best anti-AAV8 antibody retention.
  • This column was further tested by passing an IVIG composition through the column multiple times. The anti-AAV titer was then measured. Instead of a single ran we performed multiple runs on the same column to mimic the scenario in the clinic.
  • the % of retention was evaluated by anti-AAV8 ELISA. All the fractions were analyzed by ELISA and the % of retention was calculated as follows:
  • IVIG samples were prepared as follows:
  • the experimental plan was the following:
  • mice 50 male C57bl6 mice (8 weeks of age) were treated and blood/plasma was taken at the following time points : D-3, Dl, D7, D14, and D28
  • DO IV injection into tail vein: ⁇ of dog Ig (Anti-AAV8 positive, column flow through, eluted fractions from the column, naive dog serum)
  • Dl IV injection into tail vein: ⁇ of AAV8-hFIX. 5 samplings of plasma: D-3 before injection of Ig, Dl before injection of AAV8, D7, D14, and D28 before sacrifice
  • Anti-AAV8 IgG were determined as previously described (Mingozzi et al., Gene Ther 2013)
  • NABs AAV vector neutralizing antibodies
  • mice injected with dog serum containing anti-AAV8 antibodies do not show liver transduction, meaning that the dog anti-AAV8 antibodies neutralized the injected AAV8.
  • the removal of these anti-AAV8 antibodies using an AAV8 affinity column from the positive dog serum eliminated this neutralizing effect and the injected AAV8 efficiently transduced liver cells which became positive for the factor IX gene.
  • the serum from a naive dog had no inhibitory effect on liver transduction.
  • the liver cells were positive for the factor IX gene.
  • the immune-affinity column based on Sepharose (grafted with AAV8 using the NHS chemistry) is efficient and specific for removing anti-AAV8 antibodies either from human IVIG samples, from human and animal sera as well as from human plasma.
  • the best ligand (AAV8 particles) density is 1.6xl0 13 vg/column (5ml column) and it could be shown that the use of empty particles is much more efficient for anti-AAV removal than when using full particles.
  • Other AAV serotypes can also be coupled Results:
  • sepharose based chromatography gels are compressible which is not optimal when moving to large scale, we have also included such support because of several reasons: i) plasmapheresis columns, for instance, for removal of immunoglobulins are based on sepharose (e.g. Miltenyi), ii) the immobilization chemistry as proposed by GE HealthCare is rather fast in contrary to the epoxy chemistry, which is not only of advantage for the speed of the grafting procedure, but also for the integrity of the AAV particles to be grafted, and iii) in contrast to the Poros EPO support, the ligand density is known for NHS-sepharose (10 ⁇ ligand/ml gel).
  • the flow rate because impacting the dynamic binding capacity may be optimized for a given ligand density per ml of chromatography support.
  • the flow rate was varied between 0.1 and 0.5 ml/min (0.02 - 0.1 CV/min) and the results reported in Fig. 3 confirm that the reduction in the flow rate to 0.1 ml/min led to a higher anti-AAV8 removal (about 80%) in comparison to the flow rate of 0.5 ml/min (anti-AAV8 removal: about 60%).
  • each chromatographic column has a maximal dynamic capacity which was briefly evaluated by charging different volumes/quantities of Ivlg to a 5ml AAV8- NHS-sepharose column.
  • the maximal retention capacity at 0.1 ml/min could be increased from about 58% to about 81 % at a loading of 70 mg Ivlg (not shown).
  • Another way to increase the total retention (or removal) of anti-AAV8 antibodies consists in reloading of the flow-through in order to retain a supplementary amount of anti-AAV8 antibodies.
  • the flow through fractions of the first run containing 5065 ng of anti-AAV8 antibodies was reloaded onto the same column and the flow through containing about 2600 ng of antibodies, thus allowing a further retention of about 50%> of the anti-AAV8 antibodies which had not been retained in the first round.
  • the density of the ligand (here vg of grafted AAV8 particles per column volume) is of equal importance.
  • medium ligand density of about l . lxlO 13 vg/column (see above) two different ligand densities were evaluated: 9.3xlO u vg ('under-capacity') and 6.0x10 13 vg ('over-capacity').
  • AAV particles particles containing a vector genome
  • empty AAV particles virus like particles or VLPs
  • IVIG as model system is rather artificial because this is a concentrated immunoglobulin preparation with very high antibody titers not found in classical animal or human plasma samples.
  • AAV-affmity columns for the removal of anti-AAV antibodies from plasma samples, we have also evaluated different plasma samples for the removal of anti-AAV8 antibodies.
  • Figure 7A presents the retention results obtained for the loading of different volumes (range: 0.5 - 5ml) of three different plasma samples with very different anti-AAV8 titers, ranging from 1/3 over 1/270 up to 1/7290, to a 5ml AAV8-NHS-sepharose column to which 6.0x10 13 vg of AAV8 vector had been grafted.
  • Figure 7B presents the example of the removal of anti-AAV8 antibodies from a high tittering human plasma sample (1/7290). One ml of this plasma sample was loaded to the anti-AAV8 column and about 98%> of the present anti-AAV8 could be retained and only about 2% were found in the flow through.
  • AAV8 columns for the removal of anti-AAV8 antibodies from human and animal serum samples The ultimate aim is to remove anti-AAV8 antibodies from human serum, or in a model approach from animal sera.
  • 350 ⁇ 1 of human serum ('DRT with an anti-AAV8 antibody level of about 3800ng and neutralizing antibody titer of 1/316) have been loaded onto a 5ml AAV8-NHS sepharose column to which 1. lxl 0 13 vg had been grafted.
  • the passage of the human serum over the column led to the retention of 75% of anti-AAV8 antibodies (fig. 9A), a complete flow through of total human immunoglobulin (fig. 9B) and practical absence of neutralizing antibodies in all fractions (Table 2).
  • the retention was low (max: 9% for the dog serum) or non-detectable (human and macaque serum). Since the neutralizing antibody (NAB) titer is of importance and can be critical in the case the AAV vector particles are injected into patients positive for NAB the NAB titer has also to be reduced by the passage of the serum sample over the AAV8 column. In the cases of the human and the dog serum, the reduction factors were 160x and 5000x, respectively, showing clearly that the AAV8 column has also the capacity to remove specific neutralizing antibodies (Table 2). In vivo evaluation of anti-AAV8 antibody positive human plasma after treatment with an AAV8 immunoaffinity column:
  • the final proof of concept is the evaluation of the efficiency of the removal of anti-AAV8 antibodies from human plasma followed by the injection of the treated plasma with respect to the untreated plasma and the assessment of their effects on the transduction efficiency of systemically injected AAV8 one day after administration of the plasma samples to be tested.
  • the model system used was the injection of AAV8-human factor IX at a dose of 5x10 10 vg per mouse into mice which had been treated with the plasma to be tested (150 ⁇ 1 per mouse).
  • the protocol used is presented in fig. 10. As negative reference PBS was used.
  • Human plasma sample positive 1 1000 230 000ng/ 73 400 0 0 0 0
  • mice injected with anti- AAV8 positive human plasma human anti-AAV8 antibodies were detectable at significant levels up to 7 days post-injection followed by a levelling down to levels found for all other mice.
  • serum samples of mice treated with PBS or the pooled flow-through of the initially anti-AAV8 positive human plasma were negative for human anti-AAV8 antibodies.
  • NAb and IgG titers specific for AAV8 were detectable in the material eluted from the column (see Table 3), however, due to sample dilution during elution, the antibody titers measured in the elution material were lower to those in the original samples before column purification. Nevertheless, FIX transgene expression levels and vector genome copy number were maximal in mice preimmunized with the column flow through (negative for anti-AAV8 Ab), and significantly lower in animals preimmunized with the column elution material (positive for anti-AAV8 Ab). The overall results are presented in Table 3 and the detailed results with respect to anti-AAV8 antibodies found in the mouse sera at day 1 after sample injection are presented in Table 4.
  • Anti-AAV8 antibodies detected in sera of mice treated with the three different serum preparations or PBS as negative control (see table 3) one day after injection/before injection of the AAV8 vector.

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Abstract

La présente invention concerne un procédé d'élimination d'anticorps anti-AAV indésirables présents dans une composition dérivée du sang.
PCT/EP2018/055108 2017-03-02 2018-03-01 Procédé d'élimination d'anticorps anti-aav présents dans une composition dérivée du sang WO2018158397A1 (fr)

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EP4154904A1 (fr) 2018-07-17 2023-03-29 Institut National De La Sante Et De La Recherche Medicale - Inserm Compositions et procédés pour augmenter ou améliorer la transduction de vecteurs de thérapie génique et pour éliminer ou réduire des immunoglobulines
WO2021229255A1 (fr) 2020-05-14 2021-11-18 Genethon Outils et procédé de prévention d'une neutralisation des virus vaa par des anticorps
WO2021229513A1 (fr) 2020-05-14 2021-11-18 Genethon Outils et procédé pour empêcher la neutralisation d'aav par des anticorps

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